Method of gene transfer for the treatment of recessive catecholaminergic polymorphic ventricular tachycardia (CPVT)

ABSTRACT

The present invention concerns a method for the treatment of recessive Catecholaminergic Polymorphic Ventricular Tachycardia comprising delivering a gene into a cardiac cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/294,674, filed on Jun. 3, 2014, now U.S. Pat. No. 9,700,636, which isa continuation of U.S. patent application Ser. No. 13/569,490, filed onAug. 8, 2012, now U.S. Pat. No. 8,859,517, which claims the benefit ofU.S. Provisional Application No. 61/521,076, filed on Aug. 8, 2011, thecontents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention concerns a method for the treatment of recessivecatecholaminergic polymorphic ventricular tachycardia comprisingdelivering a gene into a cardiac cell.

STATE OF THE ART

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is aninherited ion channel disease characterized by high susceptibility tolife threatening arrhythmias. Two forms of the disease have beendescribed: the autosomal dominant variant and the autosomal recessivevariant. The first is associated with mutations in the cardiac ryanodinereceptor type 2 (RYR2) gene (1), while the autosomal recessive variant(2) is associated with mutations in the cardiac calsequestrin 2 (CASQ2)gene.

Clinical observations have shown that patients with the dominant form ofCPVT develop bidirectional and polymorphic ventricular tachycardia inresponse to sympathetic activation, whereas their resting ECGs areunremarkable and heart structure is preserved. The RyR2 R4496C knock-in(KI) mutant mouse model was developed to closely mimic the humanphenotype with atrial and ventricular arrhythmias during adrenergicstimulation (3). Using this model, it was demonstrated that arrhythmiasare caused by delayed after depolarizations (DADs) and triggeredactivity (TA) (4). The recessive form of CPVT is associated with a moresevere phenotype, but its pathophysiology is less well understood.Clinically, it was shown that patients with recessive CPVT have a highsusceptibility to arrhythmias triggered by emotional stress or mildexercise. Furthermore, their arrhythmias are more often polymorphicrather than bidirectional and the response to therapy is oftenincomplete. Studies performed in homozygous CASQ2^(R33Q/R33Q) mutant KImice (5) and in CASQ2 knockout (KO) (8) mice support the view thatrecessive CPVT is also associated with a much more complex phenotype atthe cellular level. CASQ2^(R33Q/R33Q) mice develop ultrastructuralrearrangements in the junctional sarcoplasmic reticulum (jSR) leading toa reduction in abundance and loss of spatial organization of crucialexcitation-contraction (EC) proteins, including triadin (Tr) and junctin(JnC) (5).

The need and importance is increasingly felt for an effective therapy incorrecting all aspects of the functional derangements observed in therecessive form of CPVT.

SUMMARY OF THE INVENTION

The present invention concerns a method for the treatment of recessiveCatecholaminergic Polymorphic Ventricular Tachycardia.

The invention will become fully clear from the following detaileddescription, given by way of a mere exemplifying and non limitingexamples, to be read with reference to the attached drawing figures.

As will be further described in the detailed description of theinvention, the method for the treatment of recessive CatecholaminergicPolymorphic Ventricular Tachycardia comprises the step of delivering aCASQ2 gene into a cardiac cell.

The authors have discovered that viral gene transfer to restore CASQ2 ishighly effective in correcting all aspects of the functionalderangements observed in the CASQ2 knock-out (KO) mice. These resultspoint at CASQ2 in the therapeutic approach based on viral gene transferof wild-type (WT) CASQ2 into CPVT recessive hearts and the developmentof early phase human clinical trials.

BRIEF DESCRIPTION OF THE DRAWINGS

The characteristics and advantages of the present invention will beapparent from the detailed description reported below, from the Examplesgiven for illustrative and non-limiting purposes, and from the annexedFIGS. 1-7.

FIG. 1.

Infection efficiency of AAV2/9. Phase contrast (left panel) and GFPsignal of the same adult myocytes isolated 20 weeks after infection withpAAV-2.1-WT-mCASQ2-IRES-GFP of a CASQ2-KO mouse. The stars areindicating the GFP positive cells corresponding to the Infected CASQ2-WTcardiomyocytes. Calibration bar=100 μm.

FIG. 2.

RealTime PCR performed on RNA extracted from adult myocytes isolatedfrom Infected WT- and WT mice. The presented results indicates ˜60% ofRNA expression of the infected WT against the 100% of the WT mice.

FIG. 3.

Western blot in isolated cardiomyocytes extracted from adult WT (C57),CASQ2-KO and Infected WT mice. AAV2/9-WT-mCASQ2 infection restores levelof expression not just of calsequestrin 2 but also of sister proteins,Tr and Jnc, other two components of the CRU. (Positive and negativesigns indicate the presence or absence of the expressed proteins).

FIG. 4.

Localization analysis of protein distribution involved in CalciumRelease Units (CRU) after infection of CASQ2-KO mice with WT-mCASQ2. Theimmunolabeling was performed with Ab anti RYR2, CASQ2, Tr and Jnclabeled in Red while in Green is possible to detect the expression ofthe reporter gene eGFP. Phase contrast in order to confirm the analysison rod-shaped cells with clear cross striations. The “star” isindicating the GFP positive cells, while the head-arrows are indicatingthe classical staining of the proteins on the CRU. Calibration bar=20μm.

FIG. 5.

Examples of triggered activity in isolated cardiomyocytes coming fromnegative GFP cells (C: not infected CASQ2-KO cells), positive GFP cells(B: infected with AAV2/9-WT-mCASQ2) and cells derived from WT mice (A).

FIG. 6.

Incidence of triggered activity in cardiomyocytes coming from negativeGFP cells (GFP-NEG, not infected CASQ2-KO cells, no=12), positive GFPcells (INF-WT, infected with AAV2/9-WT-mCASQ2, no=16) and cells derivedfrom WT mice (no=16).

FIG. 7.

ECG recording in untreated CASQ2-KO mice showing the typical pattern ofbidirectional VT upon epinephrine injection (lower panels to the left).Example of ECG recording from CASQ2-KO mice infected withAVV2/9-WT-mCASQ2 showing complete suppression of arrhythmias (Rightpanels).

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for the treatment of recessiveCatecholaminergic Polymorphic Ventricular Tachycardia.

In particular the method for the treatment of recessive CPVT accordingto the present invention comprises the step of delivering a CASQ2 geneinto a cardiac cell. The following abbreviations have been used in thepresent specification: CASQ2, calsequestrin 2; CPVT, CatecholaminergicPolymorphic Ventricular Tachycardia; CRU, calcium release unit; DAD,Delayed after depolarization; EC coupling, excitation-contractioncoupling; ECG, electrocardiogram; EP, electrophysiology; I.P.,intraperitoneal; ISO, isoproterenol; JnC, junctin; RYR2, ryanodinereceptor type 2; KO, Knock Out; Tr, triadin; WT, Wild type; UTR,untranslated repeat region; IRES, internal ribosome entry site; INF WTmouse, homozygous CASQ2 KO mouse infected with pAAV2/9-WT-mCASQ2-IRES-eGFP virus; VT, ventricular tachycardia.

In a preferred aspect the present invention relates to a method forefficient gene transfer for the treatment of recessive CPVT, wherein theCASQ2 gene is chosen from the group consisting of SEQ ID NO:1 and SEQ IDNO:2.

SEQ ID NO:1 corresponds to the coding sequence, without UTR sequences,of WT-murine CASQ2 (NM_009814.2; GI:157951699; GI:12373).

SEQ ID NO:2 corresponds to the human CASQ2 gene of NM_001232.3,GI:189011540; NM_001232.2, GI:119395726; GI: 845 and GI:209969794).

The method according to the present invention allows the correction ofthe bidirectional and polymorphic arrhythmias in patients with recessiveCPVT by a viral gene transfer method by which a gene is delivered to theheart, preferably to the cardiac myocytes and expressed, whereby thenormal and anti-arrhythmic contractile function of the heart isrestored.

Such a method of CASQ2 gene transfer surprisingly restores thephysiological levels of expression of the CASQ2 protein.

Furthermore the present inventors have found that the CASQ2 genetransfer restores the abundance and the spatial organization of crucialexcitation-contraction (EC) proteins, such as for example triadin (Tr,NP_084002.2, GeneID: 76757) and junctin (JnC, NP_075553.2, GeneID:65973).

Abnormalities in morphology of calcium release units (CRUs), geometry ofthe EC coupling apparatus and imbalance of the CASQ2/Tr/JnC complex,results in abnormal propagation of Ca²⁺ signaling.

The data already collected support the idea that viral gene transfer ofWT CASQ2 ameliorates the ultrastructural abnormalities and theassociated Ca²⁺ wave fragmentation leading to an anti-arrhythmic effect.With the recent report of the CUPID clinical trial (6) establishing thesafety of gene delivery using adeno-associated viruses in humans, theintroduction of molecular therapy in the clinics is no longer a dream.The authors believe that this investigation may provideproof-of-principle for a molecular cure for recessive CPVT, which wouldbe a highly impactful. The CASQ2 gene of SEQ ID NO:1 and SEQ ID NO:2 isin the form of plasmid DNA, wherein said plasmid DNA comprises the CASQ2gene inserted within the genome of a recombinant adenovirus.

In a preferred aspect the CASQ2 gene is inserted in a vector, preferablyinto a viral vector.

The CASQ2 gene may be advantageously inserted in the serotype 9adeno-associated viral (AAV2/9) vector.

Viral gene transfer to restore CASQ2 is highly effective in correctingall aspects of the functional derangements observed in the CASQ2 KOmice.

Moreover, based on the fact that with correction of the CASQ2 defectthere is a restoration of protein expression profiles at physiologicallevels, as well as a really strong reduction of DADs, triggered activityand a remarkable anti-arrhythmic effect in vivo, gene therapy with CASQ2corrects all the abnormalities seen in recessive CPVT models.

This supports the potential therapeutic approach based on viral genetransfer of WT CASQ2 into CPVT recessive hearts and the development ofearly phase human clinical trials.

The method according to the present invention has advantageously allowedto restore the normal intracellular Ca²⁺ storage and fluxes (between SRand cytosol) in cardiac cells, which are altered in patients withrecessive CPVT. Interestingly, similar calcium handling abnormalitieshave been detected in heart failure thus creating the rational to testthe same approach in this setting. Such a method comprising delivering aCASQ2 gene by viral gene transfer.

The observation that CASQ2 and the sister proteins Tr and JnC areinvolved in the scaffolding complex that ensures preservation of theultrastructural architecture of the jSR, will impact our understandingof the functional role of the key proteins implicated in Ca²⁺homeostasis. This notion will advance not only understanding of CPVT butin fact will also be relevant to our understanding of acquiredconditions characterized by deregulation of intracellular Ca²⁺, such asheart failure.

CASQ2 restores the intracellular Ca²⁺ deregulation in CPVT patients.

EXAMPLES Example 1

CASQ2 Gene was Cloning

The DNA of the murine WT CASQ2 gene was cloned into a bi-cistronic(pIRES) eukaryotic expression vector and sub-cloned into the multiplecloning site of pAAV2.1, serotype 9,-CMV-eGFP, containing the CMVpromoter and green fluorescent protein as reporter gene (7)

Example 2

In Vivo Infection of Cardiac Murine Myocytes Using the AAV219 Vector forEfficient WT-mCASQ2 Gene Transfer

We infected, by intraocular and intraperitoneal (I.P.) injection,neonatal (after birth, P2-P3) CASQ2 KO mice using 100 μl of serotype 9adeno-associated viral (AAV2/9) vector containing WT-mCASQ2. The micewere monitored during their development and we did not observe anydifferences in comparison with the non infected littermates. To evaluatethe infection efficiency in the mice, we performed a standard procedureof cardiac myocytes isolation by enzymatic digestion (4). The isolatedcells were plated on coverslips and observed with epifluorescentmicroscope in order to assess the presence and the level of expressionof the reporter gene, eGFP. The isolations were performed at 8, 10-12and 20-30 weeks after infection. Best results were obtained after 20weeks with a 50-60% of myocytes isolated from infected KO miceexpressing the transgene as indicated by eGFP reporter expression (FIG.1). Indeed, taking in considerations these results, the followed in vivoand in vitro experiments were achieved after 20 weeks after infection inCASQ2-KO and WT mice.

Example 3

Analysis of Expression of AAV2/9-WT-mCASQ2 in CASQ2-KO Infected Mice

While the eGFP signal detection allowed to demonstrate the correctAAV2/9-mediated delivery of the transgene (CASQ2 in our case) intocardiac myocytes, the quantification of the expression levels wascarried out through a series of in vitro assays on isolated myocytes.RealTime-PCR and immunoblot analysis provided quantification ofexpression at transcriptional and translational level of the mRNA andproteins. Quantitative RealTime PCR revealed an average of 60% (range40-80%) as compared with WT normal levels of the murine calsequestrinmRNA in the infected mice (FIG. 2). Based on this encouraging result wethan performed Immunoblot analysis in order to verify the correctexpression of the cardiac calsequestrin in adult cardiomyocytes isolatedfrom infected CASQ2-KO mice.

FIG. 3 depicts a typical Western blot experiments following theseobservations, showing that the viral transduction restored physiologicallevels of CASQ2 (FIG. 3). Furthermore, not only CASQ2, but also Tr andJnC are restored after infection with WT-mCASQ2 (FIG. 3). This stronglysupports the concept of an interdependence of expression of these threepivotal proteins.

Using confocal microscopy, we confirmed the overall cellularlocalization of the “CRU” proteins, CASQ2, RyR2, Tr and Jnc inventricular myocytes, in the WT-infected mice (FIG. 4).

Example 4

AAV2/9-WT-mCASQ2 Infection Restores the Functional Phenotype of CASQ2-KOCells

Our results suggest that CASQ2 viral gene transfer effectively leads tore-appearance of the exogenous CASQ2 in a remarkable 60% of nullbackground cells. On the basis of this result it becomes rational totest the hypothesis that AAV2/9 mediated restoration of WT calsequestrinin CPVT mice can exert an antiarrhythmic effect.

From our previous investigation we knew that CPVT arrhythmias are causedby delayed after depolarizations (DADs) and triggered activity (TA) (4)at the level of the single cardiomyocyte. Using patch clamp techniques(in current clamp mode) we analyzed the development of the DADs and/orTA in basal condition and after adrenergical stimulation.

Epifluorescence signal (from the eGFP present in our viral construct)was used to differentiate between non-infected (i.e. non-fluorescent)and infected (i.e. green fluorescent) cells and to perform comparativeassay of DAD and TA occurrence. Isolated myocytes were paced at 5 Hzfrequency at 1.5-fold the diastolic threshold and action potential wascontinuously recorded. An average of 67% of GFP negative (nonfluorescent) cells presented TA after ISO (30 nM) stimulation, while inthe same experimental condition, only 6% of the GFP positive infectedcells did. Importantly, the low incidence of TA in the infected cells isa remarkable reduction that resemble the TA registered in cellsexpressing the endogenous CASQ2 protein (FIG. 5-6).

Example 5

In Vivo Correction of the Dysfunctional Properties Observed in the CASQ2KO Mice

Thus the molecular and electrophysiological studies demonstrated acomplete normalization of the phenotype at cellular level of the CASQ2null mice after AAV2/9-WT-mCASQ2 infection, we extended our studies toin vivo characterization of the arrhythmogenic substrate in our CPVTmodel.

We used subcutaneous ECG telemeters to monitor and compare the incidenceof arrhythmias in resting conditions, during and after exercise andadrenergic induced stress.

Under resting conditions CASQ2 null mice invariably (100% incidence)presented bidirectional ventricular tachycardia while neither WT norInfected WT mice presented any ventricular arrhythmia.

We than tested the inducibility of arrhythmias in the same groups ofanimals but during adrenergic stimulation as previously described (5).Mice were injected with epinephrine (2 mg/kg) which, again, induced thetypical CPVT ventricular tachycardia in 100% of CASQ2 KO untreated mice.Conversely, no ventricular arrhythmias were detected in infected mice,just as it was found out in WT mice (Table 1 and FIG. 7).

TABLE 1 Incidence of Ventricular arrhythmias in the CASQ2-KO, WT andinfected WT mice. Resting Epinephrine Condition (2 mg/Kg) CASQ2-KO 5/55/5 INFECTED WT 0/5 0/5 WT 0/6 0/6

Viral CASQ2 gene transfer is highly effective in correcting all aspectsof the functional derangements observed in the CASQ2 KO mice (Table 2)

TABLE 2 Summary of the biological effects of AAV2/9 WT-mCASQ2 infectionon CASQ2-KO mice. Functional Results CRU proteins Ventricularrestoration Triger Activity Tachycardia Mice (Tr-Jc) (in vitro) (invivo) CASQ2-KO − + + CASQ2-WT + − − INF WT + − −Materials and MethodsAnimal Use

Animals were maintained and bred at the Charles River Laboratories inCalco, Italy, and transferred to the Maugeri Foundation forcharacterization of the phenotype. Animals were maintained and studiedaccording to the protocols approved by the Animal Care and Use facilityat the Maugeri Foundation. The adenovirus delivery was viaintaperitoneal and intra-ocular injection of 100 μl of purified virus inneonatal mice with a 25 gauge syringe in pup mice before the 3^(th) dayof born (P1-P2).

Generation of Knock-Out of CASQ2 in Mouse Model

The knock-out strain was generated by homologous recombination of thetargeting vector with 129Sv/J embryonic stem cells genome. Thelinearized targeting vector was electroporated into 129Sv/J embryonicstem cells. The clone selected with G418 and gancyclovir was injectedinto C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1females. Genotype was determined by sequencing of DNA extracted fromtail biopsy specimens (DNeasy Tissue Kit, Qiagen).

Vector Design and Production

The coding sequence, without UTR sequences, of WT-murine CASQ2(NM_009814.2) was cloned into pGEM-T-Easy vector (Promega). By enzymaticdigestion, EcoRI, the insert corresponding to the WT-mCASQ2 was excisedfrom the pGEM vector and subcloned in bis-cistronic pIRES vector (BDBiosciences, Cat. No: 631605, Clontech Palo Alto Calif., USA). Indeed,the fragment corresponding to the WT-mCASQ2 followed by the IRESsequence was subcloned via PCR amplification using specific primers(Forward: 5′-CACAGCGGCCGCACAATGAAGAGGATTTACCTGCTCATGG-3′(SEQ ID No7) andReverse 5′-CGAAGCATTAACCCTCACTAAAGGG-3′ (SEQ ID No8) containing the NotI enzymatic site. The amplicon was inserted into the adenoviral backbonevector pAAV-2.1-eGFP, serotype 9 (containing: polyA sequence type BGHand CMV promoter), provided by the Adeno-Associated Virus (AAV) vectorCore facility (Tigem, Napoli, Italy), by the enzymatic digestion withNot I. All the used plasmids were sequenced.

The AAV production was done in collaboration with the Tigem AAV Vectorcore facility(http://www.tigem.it/core-facilities/adeno-associated-virus-aav-vector-core).The AAV vectors were produced using a transient transfection of 3plasmids in 293 cells: pAd helper, pAAV rep-cap (packaging), pAAV Cis(including our insert, WT-mCASQ2-IRES, cloned in the pAAV2.1-CMV-eGFPplasmid MCS). The vectors were purified by CsCl centrifugation andundergo quality control such as Real Time PCR and Dot Blot analysis forphysical titer, or Comassie staining of SDS PAGE to evaluate thepresence and purity of capsid proteins, the infectivity (eGFP⁺ cells/ml,only for CMV-eGFP preps) and the sterility (for preps to be used inlarge animals). The service returned with a viral preparation in PBSwith a total yield >1×10¹² genome copies. All AAV stocks were frozen at−80° C. in single vial and thawed during the surgical procedure.

Isolation of Adult Mice Ventricular Myocytes

Ventricular myocytes were isolated using an established enzymaticdigestion protocol (4) from homozygous CASQ2 KO and homozygous CASQ2 KOinfected with pAAV2.1-eGFP-WT-mCASQ2 and wild-type (WT) mice (20 weeks)of either sex.

Quantitative Real-Time PCR

Real-time PCR was performed using the CFX96 Real-Time PCR DetectionSystem and analyzed using the Bio-Rad CFX Manager software package(Bio-Rad Laboratories, Inc., USA). Briefly, total RNA was purified withRneasy mini kit (Qiagen) from myocyets derived from adult CASQ2 knockoutmice and homozygous CASQ2 knockout mice infected with pAAV2.1-, serotype9, -eGFP-WT-mCASQ2 and C57/BL6 mice as WT mice. Absorbance at 260 nm(A260) was measured for each RNA sample using the NanoDrop (ND-1000)spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA). Atotal amount of 0.25 μg template RNA per reaction was taken for RT-PCRassay performed with iScript cDNA Synthesis kit (Bio-Rad Laboratories,Inc., USA). Quantitative PCR analysis was performed in optical 96-wellplates using CFX96 detection module (Bio-Rad Laboratories, Inc.). Allsamples were analyzed in triplicate with SsoFast EvaGreen Supermix usingspecific primer mix

(Forward:5′-ATGAAGAGGATTTACCTGCTCATG-3′ (SEQ ID No3), Reverse:5′-CAAGCTCCAGTACAATCTCCTTC-3′ (SEQ ID No4) and 100 ng of cDNA template.Values for threshold cycle (Ct) determination were generatedautomatically by the Bio-Rad CFX Manager software 1.5. GAPDH was used asinternal reference using the following primers: Forward:5′-GTATGACTCCACTCACGGCAA-3′ (SEQ ID No5) and Reverse:5′-GCTTCCCATTCTCGGCCTTG-3′ (SEQ ID No6).

Immunoblotting

Isolated ventricular myocytes have been processed in RIPA buffer (Thermoscientific) and total proteins extracted. Total proteins (20 μg/sample,quantified by the BCA assay) were resolved by SDS-gel electrophoresis,Novex 4-12% BisTris gradient gels using MES buffer (Invitrogen), andblotted on nitrocellulose membranes using a submarine system(Invitrogen). The membranes were probed with different antibodies:anti-CASQ2 (PA1-913, ABR), anti-Triadin (sc-33393, Santa Cruz) andanti-Junctin (a kind gift from Dr Knollmann) and anti-Actin (sc-1616-R,Santa Cruz) as reference protein. Secondary antibodies were conjugatedwith HRP (1:3000, Promega). C57/BL6 derived cells were used as positivecontrol. Specific signals were developed using the Supersignal West PicoChemiluminescent substrate (Pierce) and detected using X-ray films(Kodak).

Immunofluorescence

Isolated adult cardiomyocytes were fixed on coverslips in 4%paraformaldehyde for 10 minutes at room temperature. Coverslips werethen washed in PBS with gentle shaking. Fixed cells were permeabilizedwith 0.2% Triton X-100 in PBS for 10 minutes at room temperature. Allthe cells were kept in blocking solution (10% BSA in PBS) for 1 hour at37′C. Coverslips were then incubated for 1 hour at 37′C with a primaryantibody. The analysis was performed using following antibodies:anti-RyR2 (MA3-916, ABR), anti-CASQ2 (PA1-913, ABR), anti-Triadin(sc-33393, Santa Cruz) and anti-Junctin. After washing in PBS, cellswere incubated with Dy-Light-conjugated 594-conjugated donkeyanti-mouse/rabbit IgG secondary antibodies for 1 hour at 37° C. Thecells were washed several times in PBS and mounted on slides withmounting medium (Vectashield H-1400, Vector Laboratories, Inc, CA).Confocal microscopy was performed with a Leica TCS-SP2 digital scanningconfocal microscope equipped with a HCX PL APO 40×/numericalaperture=1.25 oil immersion objective. We used the 488-nm Argon laserline for excitation of eGFP and He/Ne laser line for excitation ofDy-Light-conjugated 594-conjugated donkey anti-mouse IgG (detected at580-630 nm). The pinhole diameter was kept at Airy 1. Images wereexported to Adobe Photoshop CS3 (Adobe Systems, Mountain View, Calif.).

Electrophyslological Recordings in Isolated Ventricular Myocytes

Cardiomyocytes were seeded on a glass bottom perfusion chamber mountedon the stage of an inverted microscope. After 5 minutes, the myocyteswere bathed with the solution containing (in mmol/L): 140 NaCl, 4 KCl, 2CaCl₂, 1 MgCl₂, 10 HEPES, and 5 glucose, pH 7.4, with NaOH. Athermostatically controlled heating ring surrounding the dish was usedto maintain the bath solution at 35° C. Transmembrane potentials wererecorded in whole cell current clamp mode using a MultiClamp 700Bamplifier (Axon Instruments). Patch electrodes were pulled fromborosilicate glass (WPI, Inc.) on a P-97 horizontal puller (SutterInstruments). The electrodes had a resistance of 2 to 3 MO when filledwith patch electrode solutions containing (in mmol/L): 120 potassiumaspartate, 20 KCl, 1 MgCl₂, 4 Na₂ATP, 0.1 GTP, 10 HEPES, 10 glucose, pH7.2, with NaOH. All signals were acquired at 10 kHz (Digidata 1322A,Axon Instruments) and analyzed with the use of personal computer runningpCLAMP version 9.2 software (Axon Instruments). Only quiescent,calcium-tolerant, rod-shaped cells with clear cross striations and aresting potential of less than or equal to −80 mV were used forelectrophysiological recordings. Myocytes were electrically stimulatedby intracellular current injection through patch electrodes usingdepolarizing pulses with duration of 3 ms and amplitude of 1.5 times theminimal current needed to evoke and action potential. The liquidjunction potential between pipette and bath solution was calculated withpCLAMP software and corrected after experiments.

ECG Monitoring, Exercise Stress Testing, and Drug Testing

ECG radiotelemetry monitors (Data Sciences International) were implantedsubcutaneously under general anaesthesia (Avertin 0.025 mg/kg). Bodytemperature was maintained at 37° C. by use of a thermally controlledheating pad (Harvard Apparatus). After 72 hours of recovery fromsurgery, phenotype characterization was performed. First, basal ECG wasrecorded for 24 hours to asses for the presence of spontaneousarrhythmias, the animals were then exercised on a treadmill untilexhaustion (15-20 min). After one day of recovery, susceptibility toadrenergically-induced arrhythmias was tested by epinephrine injection(2 mg/kg I.P.)

All animals were freely moving while ECG recordings were performed.

REFERENCES

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The invention claimed is:
 1. A method of treating recessivecatecholaminergic polymorphic ventricular tachycardia (CPVT) in a humanpatient that exhibits arrhythmia and has a deleterious mutation in thegene encoding calsequestrin 2 (CASQ2) protein, wherein said methodcomprises introducing an adeno-associated viral (AAV) vector comprisinga nucleic acid encoding a wild-type CASQ2 protein into a cardiomyocytein said patient, wherein the amino acid sequence of said CASQ2 proteinis at least 95% identical to the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2, wherein administration of said AAV vector to said patientresults in an antiarrhythmic effect on said patient, thereby treatingCPVT in the patient.
 2. The method of claim 1, wherein said AAV vectoris an AAV2/9 vector.
 3. The method of claim 1, wherein the amino acidsequence of said CASQ2 protein is at least 95% identical to the aminoacid sequence of SEQ ID NO:
 2. 4. The method of claim 1, wherein saidmutation is a homozygous mutation.
 5. The method of claim 4, whereinsaid homozygous mutation is R33Q.
 6. The method of claim 1, whereinphysiological levels of expression of CASQ2 are restored followingadministration of said AAV vector to said patient.
 7. The method ofclaim 1, wherein physiological abundance and spatial organization oftriadin and junctin are restored following administration of said AAVvector to said patient.
 8. The method of claim 1, wherein intracellularCa²⁺ storage and Ca²⁺ fluxes are restored following administration ofsaid AAV vector to said patient.
 9. The method of claim 1, wherein saidanti-arrhythmic effect is assessed by electrocardiography.